Cell-free extracts and synthesis of active hydrogenase

Abstract

Enzymatically active hydrogenase is synthesized in a cell-free reaction. The hydrogenases are synthesized in a cell-free reaction comprising a cell extract derived from microbial strains expressing at least one hydrogenase accessory protein. In some embodiments, the extracts are produced under anerobic conditions.

1. S30 extract of an  E. Coili  bacterial cell expressing at least one hydrogenase accessory protein encoding gene obtained from  Shewanella oneidensis  wherein said gene is selected from the group consisting of HydE, HydF and HydG genes. 
     
     
       2. The S30 extract of  claim 1 , wherein said bacterial cell comprises a vector encoding the Shewanella oneidensis HydE, HydF, and HydG genes. 
     
     
       3. The S30 extract of  claim 1 , wherein said extract is prepared under anaerobic conditions. 
     
     
       4. The 30 extract of  claim 1 , wherein said extract is provided in a reaction mixture suitable for cell-free polypeptide synthesis. 
     
     
       5. A method of producing enzymatically active hydrogenase protein, the method comprising:
 incubating a polynucleotide encoding a hydrogenase protein of interest in a reaction mixture comprising S30 extract according to any  claim 1  under anaerobic conditions for a period of time sufficient to synthesize said polypeptide. 
 
     
     
       6. The method according to  claim 5 , wherein said hydrogenase is an iron hydrogenase. 
     
     
       7. The method of  claim 6 , wherein said iron hydrogenase is a monomeric protein. 
     
     
       8. The method of  claim 7 , wherein said hydrogenase is selected from the group consisting of  Chlamydomonas reinhardtil  iron-hydrogenase;  Clostridium pasteunanum  hydrogenase; and  Megasphaera elsdenii hydrogenase . 
     
     
       9. The method of  claim 8 , wherein said polynucleotide encoding said hydrogenase protein of interest has been codon optimized far said S30 extract source organism. 
     
     
       10. The method of  claim 5 , wherein said synthesis is performed at a temperature from about 20° C. to about 25° C. 
     
     
       11. The method according to  claim 10 , wherein said reaction accumulates hydrogenase for at least about 24 hours.